1. Field of the Invention
The present invention relates to a genetically engineered cDNA of rat bcl-x gene and an improved protein. More particularly, it relates to a novel cDNA expressing an improved protein of Bcl-xL having higher apoptosis-inhibiting activity and cell death-inhibiting activity than the protein Bcl-xL expressed by the rat's apoptosis-inhibiting gene bcl-x. The invention also relates to materials for utilizing such cDNA in gene engineering, as well as to an improved protein of Bcl-xL expressed by the cDNA.
2. Description of the Related Art
Apoptosis is one of programmed cell death. Apoptosis is accompanied by poor contact with the surrounding cells, concentration of cytoplasm, condensation of the chromatin and nuclei associated with the endonuclease activity, fragmentation of the nuclei, formation of membrane-bounded apoptotic bodies, and phagocytosis of the apoptotic bodies by the adjacent macrophage or epithelial cells. A phenomenon that the chromosomal DNA is cleaved into DNA fragments of 180 to 200 base length by the endonuclease activity is also observed. Such phenomena have been discussed as the mechanism indicating that the apoptotic bodies are finally phagocytosed by the adjacent cells (for example, Immunology Today 7:115-119, 1986; Science 245:301-305, 1989).
As a gene controlling the apoptosis, for example, the gene bcl-2 which is an proto-oncogene found in the human follicular B cell lymphoma is known (Science 226 (4678): 1097-1099, 1984; Pro. Natl. Acad. Sci. USA 81(22): 7166-7170, 1984). Analysis of the gene structure and the transcripts or the cDNA clones have been also reported (Pro. Natl. Acad. Sci. USA 83(14): 5214-5218, 1986; Cell 47(1): 19-28, 1886). This bcl-2 gene is expressed in immune and nervous systems with high frequency. The gene product is considered to maintain homeostasis of the human immune and nervous systems by inhibiting apoptosis of the cells. Moreover, the bcl-2 gene is also considered to play an important role in morphogenesis during development since it is widely expressed particularly in the fetus
Thereafter, homologues of the gene bcl-2 were found in bovine, rat, chicken, etc., and they are collectively called the bcl-2 family.
The inventors of the present application have cloned rat bcl-x gene as a homologue of the human bcl-x gene (Cell 74(4): 597-608, 1993) belonging to the bcl-2 family (J. Biol. Chem. 271(22): 13258-13265, 1996). They have also determined the three-dimensional structure of the Bcl-xL protein expressed from the rat bcl-x gene by X-ray analysis (J. Biol. Chem. 272(44): 27886-27892, 1997).
The inventors of the present application investigated the substitutions of amino acid residue causing conformational change to enhance the anti-apoptotic activity of rat Bcl-xL. They genetically engineered cDNA of the bcl-x gene to replace a specific amino acid residue with other amino acid residue, and finally obtained the engineered cDNA whose product markedly inhibited cell death involving apoptosis.
The invention of the present application was completed based on these new findings by the inventors. The purpose of the invention is to provide the engineered cDNA that allows expression of this novel improved protein of rat Bcl-xL in cells.
Another purpose of the invention is to provide a recombinant vector containing this engineered cDNA and a cell having the recombinant vector.
Still another purpose of the invention is to provide the improved protein expressed from the above-described engineered cDNA.